Project Summary Cas9 is an RNA-guided DNA-targeting endonuclease. Its programmability facilitates two extremely useful functionalities that were previously difficult to engineer: the introduction of either a DNA double strand break or recruitment, via fusion, of a protein effector to a desired location in the genome. As a consequence, Cas9 has revolutionized both gene editing and functional interrogation of the genome. Despite this potential, a number of issues constrain Cas9's utility. Its uncontrolled nature limits both on- target endonuclease activity and the ability to promote the most desirable form of genome editing, homologous recombination. Relatedly, Cas9's natural function as an endonuclease is not necessarily congruent with that of a modular fusion protein scaffold and many effector fusions fail to elicit reliable activity. The goal of our work is therefore to create the next-generation of CRISPR-Cas proteins that enable finely-controlled genome editing activity and robust fusion protein activity. We have recently developed a series of tools, fashioned around transposon-mediated recombination, that facilitate the construction and isolation of highly engineered Cas9 fusion proteins. Using these tools, we have engineered an allosterically regulated Cas9. Here, using this and optimized variants, we propose to investigate how precise temporal control of Cas9 can improve the desired activities of on-target cutting and homologous recombination. With regards to fusion protein construction, we have preliminary data suggesting that the topology of Cas9 can be altered using circular permutation which, in principle, provides a new class of protein scaffolds for effector recruitment. We propose to systematically map Cas9's potential for circular permutation across its primary sequence and use these non-natural variants to improve one class of highly useful Cas9-effectors, transcriptional activators. Finally, in the course of our preliminary experiments, we have serendipitously discovered that circular permutation allows the construction of gated Cas9 proteins that are activated upon specific proteolytic cleavage. We propose to develop these molecules as a new class of genome editing tool and demonstrate their utility in a model system of viral infection in planta. If successful, these experiments will address several of the major challenges facing the genome editing community today: how to reduce off-target effects, increase homologous recombination activity, and exploit the programmable nature of Cas9-fusion proteins to their full extent.